A novel large-scale production system for modified basement membrane matrices using gene-swapped parietal endoderm cells.

Parietal endoderm-like cells, including Engelbreth-Holm-Swarm tumor and differentiated F9 embryonal carcinoma cells, produce huge amounts of basement membrane components, including laminin-1 (alpha1beta1gamma1). We employed a double-lox system-based gene-swapping strategy in F9 cells to replace the laminin alpha1 gene with a laminin alpha5 minigene. The gene-swapped F9 cells secreted laminin-10 (alpha5beta1gamma1) consisting of the exogenous alpha5 subunit and endogenous beta1 and gamma1 subunits on differentiation. The laminin-10 concentration in the conditioned medium exceeded 10 mg/l, which is 10-fold higher than the concentrations achieved by conventional recombinant expression systems. The gene-swapped F9 cells deposited basement membrane-like matrices containing laminin-10 on culture dishes, offering a novel microenvironment for in vitro cell manipulation.     

The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli

Abstract The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli . The dnaJ ⁻ cbpA ⁻ double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ ⁻ cbpA ⁻~ mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD , the gene that encodes the major σ ⁷⁰. The biological implications of this finding are examined and discussed.     

Use of high pressure liquid chromatography for analysis of deoxyribonucleotides in the epidermis

An analytical method for free deoxyribonucleotide (nucleoside monophosphate) in the epidermis is presented. Free nucleotides were extracted from tissue using a methylal ethanol mixture. The deoxyribonucleotides were separated using DEAE-cellulose and DBAE-cellulose. The analysis of free deoxyribonucleotides was carried out by high pressure liquid chromatography on a column of Lichrosorb-NH₂ with a single buffer of potassium phosphate. The elution order of nucleotides was CMP, AMP, UMP, IMP, GMP, and XMP. This procedure employing high pressure liquid chromatography for the detection of deoxyribonucleotides in the epidermis makes it possible to elucidate the biological characteristics and significance of DNA metabolism in normal epidermis and changes that occur in pathological conditions.